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EP1137946B1 - Utilisation du facteur de croissance endothelial vasculaire - Google Patents

Utilisation du facteur de croissance endothelial vasculaire Download PDF

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EP1137946B1
EP1137946B1 EP99959565A EP99959565A EP1137946B1 EP 1137946 B1 EP1137946 B1 EP 1137946B1 EP 99959565 A EP99959565 A EP 99959565A EP 99959565 A EP99959565 A EP 99959565A EP 1137946 B1 EP1137946 B1 EP 1137946B1
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vegf
serine
screen
phosphorylation
cells
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EP1137946A1 (fr
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James Martin Staddon
Mary Louise Morgan
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Eisai Co Ltd
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Eisai Co Ltd
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Priority claimed from GBGB9908061.6A external-priority patent/GB9908061D0/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines

Definitions

  • the present invention relates inter alia to signalling transduction pathways and the regulation of cell-cell adhesion. Such regulation is important in a variety of pathological situations.
  • the adherens junction is the site of calcium-dependent adhesion between these cells.
  • the adherens junction is composed of single-pass transmembrane adhesion molecules called the cadherins.
  • Cadherins are calcium-dependent adhesion molecules that play a key role in initiating and maintaining intercellular contact.
  • the classic cadherins are single-pass, transmembrane glycoproteins that interact homotypically with cadherins on neighbouring cells.
  • Cadherin binds directly to ⁇ - or ⁇ -catenin, proteins related to the Drosophila segment polarity gene product Armadillo, which then binds to ⁇ -catenin, a vinculin homologue, that links the complex to the actin-based cytoskeleton [see J Cell Biochem 1996 Jun 15;61(4):514-23. Cadherin-catenin complex: protein interactions and their implications for cadherin function. Aberle H, Schwartz H, Kemler R].
  • p120 another catenin, was originally discovered as a Src substrate, tyrosine phosphorylation of which may be involved in cellular transformation [see Bioessays 1997 Oct;19(10):883-91. Tyrosine phosphorylation and cadherin/catenin function. Daniel JM, Reynolds AB]. It too is a member of the Armadillo family and was subsequently realised to be associated with cadherins in both epithelial and endothelial cells. A p120 related protein p100 also exists, probably as a result of alternative splicing of the p120 gene. More details in respect of p104 and p120 can be found from WO95/13820, WO96/16170 and WO98/14186.
  • adherens junction The role of the adherens junction is to initiate and maintain cell-cell contact. Furthermore, the formation of other junctions such as the tight junction is critically dependent on the prior formation and maintenance of adherens junctions. Aberrant regulation of adherens junctions can result in metastasis, loss of contact inhibition and tissue oedema [see Pathol Res Pract 1996 Jul;192(7):694-707. Regulation of the invasion suppressor function of the cadherin/catenin complex. Vermeulen S, Van Marck V, Van Hoorde L, Van Roy F, Bracke M, Mareel M.]
  • p120 and p100 are targets for a protein kinase C (PKC) activated signalling pathway (the PKC-p120/p100 pathway) in epithelial cells.
  • PKC protein kinase C
  • protein kinase C activation resulted in decreased serine/threonine phosphorylation of p120/p100.
  • Protein kinase C activation must cause p120/p120 dephosphorylation by p120/p100 kinase inhibition or activation of the corresponding phosphatase [J Biol Chem 1997 Dec 12;272(50):31894-901. Dephosphorylation of the cadherin-associated p100/p120 proteins in response to activation of protein kinase C in epithelial cells. Ratcliffe MJ, Rubin LL, Staddon JM].
  • the PKC-p120/p100 pathway is clearly present in endothelial cells, as revealed by the use of phorbol-12,13-dibutyrate, a pharmacological activator of PKC. Endothelial cells, possess receptors for agents such as histamine and thrombin, known to be involved in the inflammatory response. It was also found that p120/p100 phosphorylation in endothelial cells is clearly regulated by these inflammatory agents (as described in WO 98/14186).
  • VE-cadherin tyrosine phosphorylation has been found to be induced by vascular endothelial growth factor (VEGF) [J Cell Science 1998 111: 1853-1865 Esser S, Lampugnani, M G, Corada M, Dejana, E and Risau W].
  • VEGF vascular endothelial growth factor
  • the present invention provides the use of VEGF to screen for a substance capable of affecting the phosphorylation state of p120 and/or p100.
  • the screen may be for a substance capable of blocking the dephosphorylation of p120 and/or p100.
  • the substance may affect the phosphorylation state of p120 and/or p100, e.g. by blocking the dephosphorylation of p120 and/or p100.
  • the dephosphorylation may be from the phosphorylated serine and/or threonine residues present in p120 and/or p100.
  • the screen is to identify a substance which is capable of affecting the phosphorylation state of p120 and/or p100.
  • a method to screen for a substance capable of affecting the phosphorylation state of p120 and/or p100, comprising the use of VEGF, is also provided.
  • the present invention provides a substance identifiable by the use of a screen as set out in the first aspect.
  • the substance may be identified by the ue of a screen as set out in the first aspect.
  • Preferred features of the first aspect of the invention apply to the second aspect.
  • the present invention provides the use of VEGF to screen for a substance capable of interfering with a VEGF-initiated pathway regulating p120/p100 serine/threonine phosphorylation.
  • the pathway may be the PKC-p120/p100 pathway.
  • the screen is to identify an inhibitor and/or a competitor and/or an activator of VEGF (in particular when the VEGF is involved in the pathway regulating p120/p100 serine/threonine phosphorylation).
  • the present invention provides an inhibitor and/or a competitor and/or an activator of VEGF identifiable by a screen as set out in the third aspect.
  • the inhibitor and/or competitor and/or activator may be identified by the screen.
  • Preferred features of the third aspect of the invention also apply to the fourth aspect.
  • the present invention provides a method to identify the phosphorylation state of p120 and/or p100 comprising identifying a mobility shift of these proteins as revealed by an immunoblotting procedure.
  • the phosphorylation state of p120 and/or p100 may be affected by VEGF.
  • the method may be for the diagnosis of a disease, which disease may involve VEGF.
  • the method may also be used in the evaluation of efficacy of a drug being used or tested to control a disease involving VEGF.
  • the present invention provides a method to identify the phosphorylation state of p120 and/or p100 comprising the use of an antibody specific for one or more of the phosphorylation sites on p120 and/or p100.
  • the method may be for the diagnosis of a disease, which disease may involve VEGF.
  • the method may also be used in the evaluation of efficacy of a drug being used or tested to control a disease involving VEGF.
  • the present invention provides a method to screen for a compound or other agent that interferes with a signalling pathway capable of being initiated by VEGF to regulate p120/p100 phosphorylation, comprising the identification of the phosphorylation state of p120 and/or p100, in the presence of the compound or the other agent, and optionally comparing the phosphorylation state with a standard.
  • the identification of the phosphorylation state may be by monitoring a band shift of p120 and/or p100.
  • the screen identifies a compound or other agent that interferes with a signalling pathway capable of being initiated by VEGF.
  • the present invention provides a compound or other agent that interferes with a signalling pathway capable of being initiated by VEGF, identifiable by a method as described in the seventh aspect of the present invention. Preferred features of the seventh aspect also apply to the eighth aspect.
  • the compound or agent may interefere with a signalling pathway that is initiated by VEGF. It may be identified by a method as set out in the seventh aspect.
  • vascular endothelial growth factor a potent and important angiogenic factor, also known as vascular permeability factor
  • VEGF vascular endothelial growth factor
  • Ferrara N Davis-Smyth T] also stimulates p120 and p100 dephosphorylation in endothelial cells.
  • VEGF 165 as well as other variants of VEGF may be used, such as amino acid sequence variants (muteins or polymorphic variants), a species of VEGF comprising additional residues, and any naturally occurring allelic and/or splice variants [Neufeld G et al, Cancer Metastasis Rev 1996 Jun 15(2) 153-8. "Similarities and differences between the vascular endothelial growth factor (VEGF) splice variants"] The p120 and p100 dephosphorylation is revealed by SDS-PAGE and immunoblotting as a band-shift (also called mobility shift), hereafter referred to as the p120/p100 band shift (as shown in Example 1).
  • a band-shift also called mobility shift
  • the invention provides a simple method of visualizing phosphorylation effects triggered by VEGF on p120/p100. Regulation of p120/p100 phosphorylation, as adherens junction-associated proteins may turn out to be important in their regulation of cell-cell adhesion, angiogenesis and tissue oedema. VEGF and therefore p120/p100 serine/threonine phosphorylation may be important in pathologies involving cancer and hypoxia.
  • VEGF is known to be an important angiogenic factor. Anti-angiogenic and therefore anti VEGF strategies are being considered as therapy for cancer and other hypoxic conditions such as retinopathy. VEGF is also a key regulator of vascular permeability in pathologies involving hypoxia. The ensuing oedema can contribute significantly to the morbidity and mortality of patients. Regulation of cadherin function may be crucial to the mode of action of VEGF.
  • the present invention reports on a method to visualize phosphorylation effects of VEGF on the cadherin-associated proteins p120 and p100. The invention could be useful in diagnosis and screening for agents interfering with VEGF initiated signalling that regulates p120/p100 phosphorylation.
  • the p120/p100 band shift can be used to diagnose biopsy samples to report on the state of p120/p100 phosphorylation. Such diagnosis may be important in making therapeutic decisions or reporting on the efficacy of therapeutic drugs.
  • the p120/p100 band shift could be used as a method to screen for compounds which interfere with VEGF-initiated signalling pathways regulating p120/p100 phosphorylation. Identification of specific phosphorylated residues within p120 or p100 affected in response to VEGF could lead to the establishment of phosphatase or kinase assays and the screening of compounds affecting such enzymes. Appropriate fusion proteins or peptides could be used as substrates.
  • a further use of the present invention is in raising or selecting antibodies.
  • Antibodies recognizing p120 or p100 in phosphorylated or dephosphorylated forms could be raised and used as described under Diagnostic Uses and Screening Uses.
  • Antibodies could be used in immunoblots, ELISAs or for immunocytochemistry of cultured cells or pathology specimens.
  • VEGF 165 was from PeproTech. Placenta growth factor (P/GF) was from R & D Systems. Phorbol-12,13-dibutyrate (PDB) was from Calbiochem-Novabiochem. Histamine was from Sigma Chemical Co. SU5416 was prepared by Tsukuba Research Laboratories, Eisai Co., Tsukuba, Japan. Collagen (Vitrogen 100) was obtained from Imperial Laboratories (Europe). [ 32 P]Phosphate (10 mCi/ml, carrier free) was from ICN-Flow Laboratories. Staphyloccocus aureus strain V8 protease (P-6306) was from Sigma.
  • Peptide-directed antibodies recognizing ⁇ - and ⁇ -catenin were provided by Kurtheimknecht (Eisai London Research Laboratories).
  • Rabbit polyclonal antibody recognizing the activated forms (phosphoThr 202 /phosphoTyr 204 ) of p42 and p44 MAP kinase was from New England BioLabs.
  • Antibodies recognizing ZO-1 and CD31 were from Zymed and Dako, respectively.
  • HRP-conjugated secondary antibodies were from Amersham.
  • fluorochrome-conjugated (Cy3 or fluorescein) secondary antibodies and the rabbit anti-mouse IgG were from Jackson ImmunoResearch Labs.
  • Human umbilical vein endothelial cells pooled donors, were from BioWhittaker and cultured in EGM on collagen-coated plastic (Falcon) according to the supplier's instructions. The cells were cultured in medium in the presence of 100 U/ml penicillin and 100 ⁇ g/ml streptomycin at 37°C in humidified air containing 10% CO 2 . Routinely, stocks were maintained in 75 cm 2 flasks and split at a 1:3 ratio when almost confluent. For experimental use, cells were seeded in appropriate plasticware such that confluence was established within 2-3 days and the cells were usually used 5-6 days after seeding.
  • [ 32 P]Phosphate labelling was performed using Puck's Saline A (P-2917; Sigma) supplemented with 10 mM Hepes (Gibco), 2 mM glutamine (Gibco), 1 mM CaCl 2 , 1 mM MgSO 4 and 0.2% (v/v) foetal calf serum. Cultures were rinsed twice with medium and then incubated with the same medium containing 100 ⁇ Ci/ml of [ 32 P]phosphate. Labelling was for 2 hours at 37°C in humidified air with ambient CO 2 .
  • the gels were then equilibrated for 30 minutes in buffer at 4°C containing 48 mM Tris, 39 mM glycine, 0.03% SDS (w/v) and 20% methanol (v/v), and electrotransferred to nitrocellulose filters (Hybond ECL, Amersham) at 4°C. After Ponceau S staining, the filters were blocked in PBS containing 1% (w/v) BSA and 0.05% (v/v) Tween-20. The filters were then incubated with primary antibody, rinsed and immunoreactive bands were detected using appropriate HRP-conjugated secondary antibody and chemiluminescence (ECL, Amersham).
  • the lysates were centrifuged at 14,000 x g for 20 minutes. The supernatant was precleared with protein A-Sepharose (Pharmacia) for 1 hour and then incubated with primary antibody for 1 hour followed by a further 1 hour with protein A-Sepharose together with rabbit anti-mouse antibodies. After five washes in lysis buffer, immune complexes were dissociated by addition of Laemmli sample buffer followed by heating at 100°C for 5 minutes. Protein analysis was by SDS-PAGE and immunoblotting as described above.
  • p120/p100-specific and phosphotyrosine (PY20) protein immunoprecipitations the cells were lysed in boiling SDSSB (1% SDS, 25 mM Hepes, 2 mM EDTA, 0.1 M NaCl, 25 mM NaF, 1 mM vanadate, pH 7.6). The lysate was then collected, heated at 100°C for 5 minutes, cooled and diluted with four volumes of ice-cold TX buffer that contained 2.9% instead of 1% Triton X-100. Immunoprecipitation was performed as described for TX buffer extractions.
  • the filter was then probed with antibody recognizing p120 and p100 to quantitate protein levels, as described.
  • preflashed film was used for both the autoradiography and luminography.
  • proteins were transferred to Immobilon P (Millipore).
  • the area of filter containing p120 or p100 was detected by autoradiography, excised, and protein was acid hydrolysed to release phosphoaminoacids. These were then separated in two dimensions by high voltage electrophoresis at pH 1.9 and pH 3.5 and detected by autoradiography (Boyle et al., 1991).
  • proteolytic mapping Cleveland, D. W., Fischer, S. G., Kirschner, M. W.
  • Confluent monolayers of cells were fixed at room temperature for 15 minutes in 3% paraformaldehyde made up in PBS. Fixed cells were rinsed and then permeabilized by incubation with 0.5% Triton X-100 in PBS for 30 minutes. All antibodies were diluted in PBS containing 10% FCS, 0.1 M lysine, 0.3% Triton-X-100 and 0.02% azide. Incubation with antibody recognizing junction-associated protein was at 4°C overnight followed by incubation with mouse or rabbit anti-phosphotyrosine antibody for 2 hours at room temperature. Secondary antibodies recognizing mouse and rabbit IgG conjugated to Cy3 or fluorescein, respectively, were applied together for 1 hour at room temperature. After rinsing, the samples were mounted under Citifluor and viewed with epifluorescence on a Nikon Eclipse E800 microscope. Photographs were taken using Kodak T-MAX film (400 ASA).
  • Human umbilical vein endothelial cells were incubated in the absence (Control - lane labelled C) or presence of 2.6 nM VEGF for 5 minutes. Incubations were performed in triplicate and analyzed in parallel. The cells were extracted in Laemmli sample buffer and proteins were resolved by SDS-PAGE. Following transfer to nitrocellulose, the filters were probed with antibody recognizing p120 and p100 (see Fig. 1A). p120 (migration indicated by the open circle) and p100 (migration indicated by the closed circle) from untreated cells migrate as broad bands. Those from VEGF treated cells (lane labelled V) migrate as faster, tighter bands.
  • VEGF vascular endothelial growth factor
  • VEGF acted very rapidly to trigger the increase in mobility of p120 and p100, effects were observed within 2 minutes and sustained for up to 30 minutes (Fig. 2A). After 60 minutes of incubation, the effect was less apparent (Fig. 2A). The kinetics paralleled those of the increase in amount of activated MAP kinase (Fig. 2B).
  • VEGF is known to bind to two receptors, VEGFR-1 and VEGFR-2.
  • P l GF placenta derived growth factor
  • VEGFR-1-specific ligand a VEGFR-1-specific ligand
  • the cells were preincubated for 30 minutes in the absence (-) or presence(+) of 5 ⁇ M of the VEGFR-2 inhibitor SU5416. The cells were then treated for 5 minutes with either 2.6 nM VEGF (V) or 10 ⁇ M histamine (H). In all cases, cell extracts were prepared and p120 (O) and p100 (•) were detected by immunoblotting following SDS-PAGE, Figures 4A and 4C. The same filter was also probed with antibody recognizing the activated forms of p42 and p44 MAP kinase (arrowheads), Figures 4B and 4D. Equal loading of lanes was verified as in Fig. 1.
  • VEGFR-2 VEGFR-2 specific inhibitor SU5416 (Sun, L., Tran, N., Tang, F., App, H., Hirth, P., McMahon, G. and Tang, C.
  • SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res. 59 99-106) blocked the ability of VEGF to stimulate an increase in p120/p100 mobility (Fig. 4C) and activation of MAP kinase (Fig. 4D). In contrast, SU5416 did not block the ability of histamine to stimulate a mobility shift in p120/p100 or to activate MAP kinase (Figs. 4C,D), indicating selectivity in its mode of action.
  • Flk-1/KDR vascular endothelial growth factor receptor
  • phosphorylation analysis was performed.
  • the cells were metabolically labelled with [ 32 P]phosphate, stimulated as required, lysed in buffer designed to preserve the phosphorylation state of proteins, immunoprecipitated with antibody recognizing p120 and p100 and then analyzed by SDS-PAGE, autoradiography and immunoblotting. This approach allows quantitation of phosphate per amount of protein.
  • HUVECs Confluent monolayers of HUVECs were metabolically labelled with [ 32 P]phosphate. The cells were then treated for 5 minutes with vehicle (lanes labelled C), 10 ⁇ M histamine (lanes labelled H), 200 nM PDB (lanes labelled P) or 2.6 nM VEGF (lanes labelled V). Following lysis in TDS buffer, p120 and p100 were immunoprecipitated, resolved by SDS-PAGE and transferred to nitrocellulose. An autoradiogram of the filter was taken to detect [ 32 P]phosphate incorporation into p120 (O) and p100 (•), Figure 5A, and the filter was then probed with antibody recognizing p120 and p100 to detect protein (Figure 5B).
  • p120 and p100 were detected as phosphoproteins in control cells and the [32P]phosphate:protein was determined (Fig. 5A).
  • the autoradiogram showed that the [ 32 P]phosphate signal was less and the film density was similar to that of p120/p100 from control cells (Fig. 5A).
  • Examination of the immunoblot of the same filter showed that VEGF caused an increase in p120/p100 mobility and an increase in film density of the ECL signal. Quantitation by densitometry revealed that VEGF stimulated dephosphorylation of p120 and p100 (Fig.
  • HUVECs were metabolically labelled with [ 32 P]phosphate, stimulated as required and phosphorylation site analysis was performed by Cleveland mapping, a procedure involving limited proteolytic digestion.
  • HUVECs Confluent monolayers of HUVECs were metabolically labelled with [ 32 P]phosphate. The cells were then treated for 5 minutes with vehicle (C), 10 ⁇ M histamine (H), 200 nM PDB (P) or 2.6 nM VEGF (V). Following lysis in TDS buffer, p120 and p100 were immunoprecipitated and resolved by SDS-PAGE. The gel was briefly fixed and an autoradiogram taken to localize bands corresponding to p120 and p100. These were excised from the gel partially digested with V8 protease and the digestion products were analyzed by SDS-PAGE. The labelling of digestion products was detected again by autoradiography. It was confirmed by immunoblotting that the amounts of p120 and p100 in the initial immunoprecipitates were equal.
  • VEGF and histamine produced effects on p120/p100 (Fig. 7A) and MAP kinase (Fig. 7B).
  • Pervanadate (Pv) did not affect the mobility of p120/p100 (Fig. 7A) but did lead to activation of MAP kinase (Fig. 7B).
  • the levels of ⁇ catenin were found to be similar in all extracts (Fig. 7C).
  • Phosphoaminoacid analysis of p120/p100 was also performed.
  • p120 and p100 were phosphorylated on mainly serine and, to a lesser extent, threonine residues (Fig. 7G).
  • Confluent monolayers of HUVECs were metabolically labelled with [ 32 P]phosphate and received either vehicle (C), 10 ⁇ M histamine (H), 200 nM PDB (P) or 2.6 nM VEGF (V) for 5 minutes.
  • C vehicle
  • H 10 ⁇ M histamine
  • P 200 nM PDB
  • V 2.6 nM VEGF
  • HUVECs were either treated with vehicle (Control) or 2.6 nM VEGF. After 5 minutes, the cells were fixed and then stained. In parallel, cells were also extracted and analyzed for p120/p100 by immunoblotting to confirm that VEGF had in fact stimulated a p120/p100 shift.
  • Panel A shows the results of colabelling with mouse antibody recognizing ⁇ -catenin and rabbit antibody recognizing phosphotyrosine (pY). The localization of ⁇ -catenin was not affected by VEGF treatment.
  • Panel B shows the results of colabelling with mouse antibody recognizing vinculin and rabbit antibody recognizing phosphotyrosine (pY).
  • VEGF treatment resulted in the appearance of vinculin-positive striations close to regions of cell-cell contact; these striations colabelled with phosphotyrosine antibody.
  • phosphotyrosine staining was competed by phosphotyrosine but not by phosphoserine or phosphothreonine. All images were from similarly timed exposures. Bar, 20 ⁇ m.
  • Cadherins associate via their cytoplasmic tail with the catenins, including p120 and p100.
  • the cells were lysed under detergent conditions designed to preserve association of the complex. Lysates from control or VEGF-stimulated (2.6 nM, 5 minutes) cells were immunoprecipitated with antibodies recognizing both p120 and p100 or ⁇ -catenin. The immunoprecipitates were analyzed by immunoblotting for the content of VE-cadherin, N-cadherin, ⁇ , ⁇ and ⁇ catenin, p120 and p100.
  • VEGF treatment although increasing the electrophoretic mobility of p120 and p100, had no effect on association of any members of the complex (results not shown). Furthermore, immunocytochemical analysis also did not reveal any apparent effects on localization of ⁇ -catenin (Fig. 8) or p120/p100 (result not shown) to adherens junctions following VEGF treatment.

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Claims (12)

  1. Utilisation du VEGF pour cribler in vitro une substance pouvant affecter l'état de phosphorylation des sérines et/ou thréonines de p120 et/ou p100.
  2. Utilisation du VEGF selon la revendication 1, dans laquelle on crible une substance pouvant bloquer la déphosphorylation des sérines et/ou thréonines de p120 et/ou p100.
  3. Utilisation selon la revendication 2, dans laquelle la déphosphorylation a lieu à partir des résidus phosphorylés de sérine et/ou de thréonine présents dans p120 et/ou p100.
  4. Utilisation d'un criblage selon l'une quelconque des revendications 1 à 3, pour identifier une substance qui peut affecter l'état de phosphorylation des sérines et/ou thréonines de p120 et/ou p100.
  5. Utilisation du VEGF pour cribler in vitro une substance pouvant interférer avec une voie initiée par le VEGF qui régule la phosphorylation des sérine/thréonine des p120/p100.
  6. Utilisation du VEGF selon la revendication 5, dans laquelle la voie est la voie PKC-p120/p100.
  7. Utilisation du VEGF selon la revendication 5 ou la revendication 6, dans laquelle le criblage sert à identifier un inhibiteur et/ou un compétiteur et/ou un activateur du VEGF.
  8. Procédé d'identification in vitro de l'état de phosphorylation des sérines et/ou thréonines de p120 et/ou p100, comprenant l'utilisation d'un anticorps spécifique à l'un ou à plusieurs des sites de phosphorylation sérine et/ou thréonine sur p120 et/ou p100, dans le diagnostic d'une maladie impliquant le VEGF.
  9. Procédé d'identification in vitro de l'état de phosphorylation des sérines et/ou thréonines de p120 et/ou p100, comprenant l'utilisation d'un anticorps spécifique à l'un ou à plusieurs des sites de phosphorylation sérine et/ou thréonine sur p120 et/ou p100, dans l'évaluation de l'efficacité d'un médicament que l'on utilise ou que l'on évalue pour contrôler les maladies impliquant le VEGF.
  10. Procédé de criblage in vitro d'un composé ou d'un autre agent qui interfère avec une voie de signalisation pouvant être initiée par le VEGF pour réguler la phosphorylation de p120/p100, comprenant l'identification de l'état de phosphorylation des sérines et/ou thréonines de p120 et/ou p100, en présence du composé ou de l'autre agent, et comprenant éventuellement la comparaison de l'état de phosphorylation avec un standard.
  11. Procédé selon la revendication 10, dans lequel l'identification de l'état de phosphorylation des sérines et/ou thréonines se fait en contrôlant un déplacement des bandes de p120 et/ou p100.
  12. Procédé selon la revendication 10 ou 11, dans lequel le criblage identifie un composé ou un autre agent qui interfère avec une voie de signalisation pouvant être initiée par le VEGF.
EP99959565A 1998-12-11 1999-12-10 Utilisation du facteur de croissance endothelial vasculaire Expired - Lifetime EP1137946B1 (fr)

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Application Number Priority Date Filing Date Title
GB9827407 1998-12-11
GBGB9827407.9A GB9827407D0 (en) 1998-12-11 1998-12-11 Methods
GB9908061 1999-04-08
GBGB9908061.6A GB9908061D0 (en) 1999-04-08 1999-04-08 Methods
PCT/GB1999/004162 WO2000036421A1 (fr) 1998-12-11 1999-12-10 Utilisation du facteur de croissance endothelial vasculaire

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EP1137946B1 true EP1137946B1 (fr) 2005-10-19

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US5155031A (en) * 1990-06-07 1992-10-13 Posner Barry I Use of pervanadate as an inhibitor of phosphotyrosine phosphatase
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JP2002532720A (ja) 2002-10-02
US20030134334A1 (en) 2003-07-17
ATE307339T1 (de) 2005-11-15
WO2000036421A1 (fr) 2000-06-22
WO2000036421A8 (fr) 2000-12-21
DE69927845D1 (de) 2005-11-24
US20020037544A1 (en) 2002-03-28
US20030129662A1 (en) 2003-07-10

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